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Home , Chromium toxicity

Global Veterinaria 16 (4): 323-329, 2016 ISSN 1992-6197 © IDOSI Publications, 2016 DOI: 10.5829/idosi.gv.2016.16.04.10332

Protective Effect of Melatonin Against -Induced Hepatotoxic and Genotoxic Effect in Albino Rats

Emad A. Hashish and Shimaa A. Elgaml

Department of Clinical Pathology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Sharkyia, Egypt

Abstract: Chromium is a widespread environmental waste. It is an industrial contaminant with teratogenic, mutagenic and carcinogenic effects on animals and human. This study was carried out to evaluate the potential protective effect of melatonin on the hepatotoxicity and genotoxicity generated by potassium dichromate

(K2 Cr 27 O ) in albino rats. Rats were divided into four groups; control, melatonin (10 mg/kg b.wt.), melatonin

pretreated with single S/C injection of K2 Cr 27 O (15 mg/kg b.wt.) and potassium dichromate treated group.

Rats were sacrificed 24 h after K2 Cr 27 O treatment. K2 Cr 27 O treated rats showed significant (P<0.05) increase in the hepatic marker enzymes activity (aspartate aminotransferase-AST and alanine aminotransferase-ALT) and serum bilirubin (total and direct) was detected. Significant (P<0.05) decrease in the serum total protein and

albumin was observed after K2 Cr 27 O treatment. Meanwhile, serum globulin, A/G ratio and indirect bilirubin showed no significant change. Hepatic DNA damage was observed using comet assay. The histological alterations confirmed the previous results. Melatonin pretreated rats showed an amelioration of the

adverse effects of K2 Cr 27 O . An improvement in the serum hepatic enzymes (AST and ALT), proteinogram (total protein and albumin) and bilirubin (total and direct) was observed. Melatonin pre-treatment showed significant improvement in the hepatic DNA and the histopathological picture. It could be concluded that potassium dichromate is hepatotoxic and genotoxic. Melatonin has a potential protective effect to reverse

the DNA damage and has the ability to improve the hepatic function associated with K2 Cr 27 O intoxication.

Key words: Chromium Hepatotoxicity Genotoxicity Melatonin

INTRODUCTION activities of enzymes involved in the antioxidative defense [3]. It protects the in several models of liver injury via Chromium is a transition which can be found in inhibiting the oxidative and the nitrosative damage [4]. many compounds of Earth's crust. It is ranked the 21st in Its protection against the oxidative damage is enhanced elemental abundance. Chromium comes from several by its amphiphilic nature. The melatonin molecule is anthropogenic sources such as chemical, metallurgical readily access to all compartments including and refractory industry [1]. It is responsible for various the nucleus [5]. Melatonin stimulates the immune health hazards including cancers, dermatitis, damage to response and exhibits immunomodulatory effects [6]. the liver and , infertility in both males and females, The immunomodulatory properties include the inhibition defects in embryo and developmental problems in young of the expression of cyclooxygenase 2 (COX-2) and children [2]. inducible nitric oxide synthase (iNOS) to exert marked Melatonin (N-acetyl-5-methoxytryptamine) is the anti-inflammatory effect [7]. It also mitigates the oxidative main product of the pineal gland in both stress and the vital organ damage induced by heavy animals and human. It plays a major role in the and snake toxicity [8, 9]. neuroimmuno-endocrine system. Melatonin participates Intensive research over the last two decades has in many vital physiological functions such as reported the beneficial protective effect of melatonin in a anti-inflammatory and a broad spectrum antioxidant. multitude of pathological processes. It is considered as an It decreases the level of free by stimulating the immunostimulant for both humeral and cellular immunity

Corresponding Author: Shimaa A Elgaml, Clinical Pathology Department, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt, 44511. Cell: +201009168681, Fax: +20552283683. E-mail: [email protected]. 323 Global Veterinaria, 16 (4): 223-329, 2016

[10]. No previous studies reported the protective effect of flow smoothly into the tubes, left at room temperature for melatonin against chromium induced hepatoxicity. 2 hours to clot then centrifuged at 3000 rpm for 15 min. Therefore, in the present investigation, an attempt was The clear supernatant serum was collected using sterile made to evaluate the protective effect of melatonin on Pasteur pipettes. Serum was transferred to sterile, potassium dichromate mediated hepatotoxicity in order to dry labeled eppendorf tubes for chemical analysis. study the role of melatonin in the amelioration of the Liver from all groups was removed, washed with saline chromium intoxication. and cut into two parts, one part was preserved at -20°C for comet assay and the other part was preserved in MATERIALS AND METHODS 10% neutral-buffered formalin for the histological investigation. Chemicals: Melatonin was obtained from Sigma-Aldrich (product number M5250). Melatonin was administered as Biochemical Analysis: All biochemical analysis was single intraperitoneal injection 10 mg/kg b.wt., daily for 10 calorimetrically measured using commercial kits provided successive days [11] before induction of . by bioMérieux, France. The analysis was done using a

Potassium dichromate (K2 Cr 27 O ) was obtained from spectrophotometer (5010 v5C, RIELE GmbH, Berlin, Sigma-Aldrich (product number 207802). It was used to Germany). induce acute toxicity. Rats were given a single subcutaneous (S/C) injection of K2 Cr 27 O at a dose of Serum Enzymes: Serum aspartate aminotransferase (AST) 15 mg/kg b.wt. [12]. and serum alanine aminotransferase (ALT) activity were determined [13]. Experimental Animals: Thirty two female albino rats weighing 150-200 g were obtained from the Laboratory Serum Proteins: Serum total protein was colometrically Animal House, Helwan, Egypt. Throughout the measured [14]. Serum albumin was colometrically experiment, all animals were housed in clean hygienic measured [15]. Serum globulin level was determined by metal cages inside environmentally controlled room and subtracting the albumin from the total proteins. kept under a uniform laboratory condition. All rats were Albumin/Globulin ratio (A/G ratio) was measured using administered balanced ration and water free from albumin and globulin values. . The food and were allowed ad.libitum throughout the experimental period. Rats were Serum Bilirubin: Serum total and direct bilirubin were maintained under standardized hygienic conditions measured [16]. The indirect bilirubin was measured (12:12 h light/dark cycles, the temperature was 24±2 °C through subtracting the obtained direct bilirubin from the and a minimum relative humidity of 45%) throughout total bilirubin. the experimental period. All rats were acclimatized for 2 weeks before starting the experiment. Comet Assay for Detection of Hepatic DNA Damage: The study was complied with the Animal Welfare Act. The alkaline comet assay was done using single cell gel The National Research Council Committee guidelines were electrophoresis according to [17]. Briefly, liver tissue (1 g) followed. was crushed and transferred to 1 mL ice-cold PBS, pH 7.4 and homogenized with a Potter-Elvehjem Experimental Design: Rats were divided into four groups; type-homogenizer (Ikemoto Scientific Technology, Tokyo, Group I, served as control. Group II, melatonin was IP Japan). The suspension was centrifuged at 500 RPM/ 5 administered daily at a dose of 10 mg/kg b.wt for 10 days. min; the supernatant was aspirated and repeat Group III, melatonin pretreated for 10 days then centrifugation for 3 times in PBS. The last suspension was subcutaneous (S/C) injected with a single dose of K2 Cr 27 O centrifuged at 3000 RPM/10 min to sediment hepatic cells.

15 mg/kg b.wt. Group IV, was S/C administered K2 Cr 27 O , An aliquot of the cell suspension was mixed with 0.5% 15 mg/kg b.wt. dissolved in distilled water. The samples low melting agarose then spread using a cover slide were collected 24 h post KCr2 27 O injection. on pre-coated slides with 1% normal-melting agarose, freeze for 2min then remove the cover slide. Then slides Blood and Liver Samples: Rats from all groups were were immersed in a lysis buffer over night. The slides were autopsied under light ether anesthesia 24 h post K2 Cr 27 O transferred to electrophoresis buffer in electrophoresis injection. The blood was drawn from the retro-orbital chamber. The current of electrophorsis was adjusted to venous sinus using capillary tubes. Blood was allowed to 300mA for 30 min.

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Slides were stained with with 1% ethidium bromide at RESULTS 4 °C, then examined under a fluorescence microscope. About 50 to 100 random cells were selected and analyzed The Activity of the Hepatic Marker Enzymes (AST and per each sample and analyzed by Comet ScoreTM software ALT) of all Treated TM Rats was Evaluated: Group IV to calibrate the comet parameters. The software was used (K2 Cr 27 O treated rats) showed a significant (P<0.05) to evaluate the quantitative and qualitative DNA damage increase in the activities of the hepatic enzymes in the cells by measuring the tail length, tail DNA comparing with the control. Group II (melatonin treated) percentage and tail moment. showed a non significant change in the activities of these enzymes comparing with the control. This indicates the Histopathological Study: Rats were anesthetized with safe use of melatonin as a natural product. Group III diethyl ether and sacrificed. A portion of liver (melatonin pretreated) showed significant (P<0.05) tissue from all groups was collected and immediately decrease in the activity of these hepatic enzymes in the prepared for hisopathological examination [18]. serum when compared to the group IV (K2 Cr 27 O treated). Simply, the specimen labeled and fixed in 10 % neutral The ameliorative effect of the melatonin pre-treatment was pronounced (Figure 1). buffered formalin solution overnight. Fixed tissues were processed after washing and preserved in 70 % Assessment of Proteinogram Level of all Treated Rats: ethanol, dehydrated in a series of graded concentrations Group IV (K Cr O treated rats) showed a significant of ethyl and cleared in xylene. Tissue were 2 27 (P<0.05) decrease in the level of serum total protein embedded in paraffin waxes (melted paraffin at and albumin comparing with the control. Group II 55–60 °C), casting and cutting at 4-5 µm thickness. (melatonin treated) showed a non significant change These sections were placed on top of glass slides, when compared with the control. Group III routinely stained with Hematoxylin and Eosin (melatonin pretreated) showed significant (P<0.05) (H and E) to be examined and visualized under the increase in the level of serum total protein and albumin microscope. when compared to the group IV (K2 Cr 27 O treated). The serum globulin level and A/G ratio showed Statistical Analysis: System software package was used a non significant change in all treated groups to analyze the data by one-way analysis of variance, (Figure 2). ANOVA [19]. All data showed a normal distribution and passed equal variance testing. The Data were expressed The Level of Serum Bilirubin: Group IV (K2 Cr 27 O treated as the means ± standard error (SE). Significant differences rats) showed a significant (P<0.05) increase in the level of between means were determined at a level of (P<0.05) by serum bilirubin (total and indirect) comparing with the post-hoc Duncan’s test for comparison between different control. Groups II and III showed a non significant experimental groups. change in the serum level of total and direct bilirubin

Fig. 1: Effect of pretreatment of melatonin on the hepatic serum enzymes (AST and ALT). Melatonin treated and pretreated rats, showed a non significant change comparing with the control. Group IV showed a significant (P<0.05) increase in the AST and ALT level comparing with the control. Bars with different letters are expressed at (P < 0.05) in means ± SE.

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Fig. 2: Effect of pretreatment of melatonin on proteinogram. Melatonin treated and pretreated rats, showed a non significant change in the proteinogram comparing with the control. Group IV showed a significant (P<0.05) decrease in the level of total protein and albumin comparing with the control. Bars with different letters are expressed at (P < 0.05) in means ± SE.

Fig. 3: Effect of pretreatment of melatonin on serum bilirubin level. Melatonin treated and pretreated rats, showed a non significant change in serum bilirubin (total, direct and indirect) comparing with the control. Group IV showed a significant (P<0.05) increase in the level of total and direct bilirubin comparing with the control. Bars with different letters are expressed at (P < 0.05) in means ± SE. comparing with the control. The level of the serum indirect bilirubin showed a non significant change in all treated groups (Figure 3).

Effect of Melatonin on Cr-induced-DNA Damage in Liver Was Determined: Control and melatonin treated rats showed normal hepatic cells as no hepatic DNA damage was noticed (Figure 4 A and B). Meanwhile, melatonin pre-treated rats showed an improvement in the comet parameters (tail percentage and length, beside the DNA tail percentage and moment) (Figure 4C). Pre-treatment Fig. 4: Hepatic cell images of the comet assay. A) with melatonin restored the DNA damage parameters in Control. B) Melatonin treated rats. C) Melatonin the liver to near control. Cr induced an increase in DNA pretreated group. D) Chromium treated group. damage as indicated by a significant increase in the tail percentage and length beside an increase in the tail Histopathological Examination of the Hepatic Tissue: moment in the liver of the Cr-treated rats, group IV with Normal liver structure was observed in the control group marked damaging effect on the DNA (Figure 4D). (Figure 5A). Group II, melatonin treated, the hepatic tissue

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Fig. 5: Histopathological finding of the hepatic tissue, HE x400. A) Control group showed normal hepatic architecture. B) Melatonin treated, showing mild proliferation of the hepatic Kupffer cells (arrow). C) Melatonin pretreatment, showing swollen hepatocytes with intact nuclei (arrows). D) Chromium treated group showing congested central vein (arrow) and dilation of the hepatic sinusoids (arrow head). E) Chromium treated group, showing moderate mononuclear cells infiltration in the portal area (arrow). appeared normal with mild proliferation of the hepatic disturbance in their biosynthesis due to the liver Kupffer cells (Figure 5B). The liver in group III, dysfunction with alteration in the permeability of the melatonin-pretreated, showed nearly normal architecture hepatic membrane [21]. Our results were confirmed by the with moderate degenerative changes of hepatocytes histopathological observations. Significant decrease in which appeared swollen with intact nuclei (Figure 5C). the serum AST and ALT levels was observed In addition, moderate congestion was seen in the central with melatonin pre-treatment. Protective effects of vein. Severe liver damage was detected in group IV, melatonin in decreasing the level of these hepatic Cr treated, with congestion of the hepatic blood vessels markers correlated with the histological findings. and dilation of hepatic sinusoids was seen throughout Our results are in accordance with A'maal et al. [22] the hepatic tissue (Figure 5D). The portal area where similar decrease was reported in these hepatic showed moderate mononuclear cells infiltration marker in rats administered melatonin as protective (Figure 5E). agent against chlorpromazine-induced liver disease in rats. DISCUSSION Serum total protein, albumin and globulin reflect the damage in the hepatocytes. In the present study, Melatonin has a number of beneficial effects in the serum total protein and albumin were significantly treatment of various types of diseases. This study decreased in Cr-treated rats. These results demonstrate investigated the protective effect of melatonin that Cr toxicity was associated with hepatotoxicity. against Cr-induced hepatotoxicity. The transaminases This may be due to the impairment in their synthesis (AST and ALT) are considered the most sensitive or poor liver function [21]. Similar results were observed biomarkers to evaluate the extent of the toxicity [23, 24]. On the other hand, a significant increase in the and the cellular damage. The hepatotoxicity of Cr resulted level of serum total protein and albumin was observed in in increase the activity of these hepatic enzymes rats pretreated with melatonin. Our results were (AST and ALT). Because these enzymes are located in the confirmed with the histopathological investigation. In the hepatic cytoplasm and after cellular damage they are present study, the hepatic histoarchitecture of the released in the blood stream [20]. Chromium (Cr) might be chromium-treated rats resulted in congestion of the responsible for leakage of these enzymes from the liver hepatic blood vessels, dilation of hepatic sinusoids and cytosol into the circulation or it might be due to mononuclear cells infiltration throughout the hepatic

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